Engineering of functional supramacromolecular complexes of proteins (enzymes) using reversed micelles as matrix microreactors

The size of the inner water cavity of reversed micelles formedin a triple system ‘water-surfactant-organic solvent’can be widely varied by changing the degree of surfactant hydration.This gives grounds to use reversed micelles as matrix microreactorsfor the design of supramolecular complexes of proteins. Usingultracentrifugation analysis, it has been demonstrated thatthe oligomeric composition of various enzymes (ketoglutaratedehydrogenase, alkaline phosphatase, lactic dehydrogenase, glyceraldehyde-3-phosphatedehydrogenase) solubilized in reversed micelles of Aerosol OT[sodium bis(2-ethylehexyl)sulfosuccinate] in octane changesupon variation of the degree of hydration. An oligomeric complexforms under conditions when the radius of the micelle innercavity is big enough to incorporate this complex as a whole.At lower degrees of hydration the micelles ‘uncouple’such complexes to their components. The catalytic propertiesof various oligomeric complexes have been studied. Possibilitiesof using reversed micelles for the separation of subunits ofoligomeric enzymes under non-denaturating conditions have beendemonstrated. In particular, the isolated subunits of alkalinephosphatase, lactic dehydrogenase and glyceralde-hyde-3-phosphatedehydrogenase have been found to be active in Aerosol OT reversedmicelles. The dependences of the catalytic activity of oligomericenzymes represent saw-like curves. The maxima of the catalyticactivity observed at these curves relate to the functioningof various oligomeric forms of an enzyme. The radii of the micelleinner cavity under conditions when these maxima are observedcorrelate with the linear dimensions of the enzyme oligomericforms. Correlation of the position of a maximum with the shapeof an oligomeric complex is discussed.     

Print your atomic force microscope

Progress in scanning probe microscopy profited from a flourishing multitude of new instrument designs, which lead to novel imaging modes and as a consequence to innovative microscopes. Often these designs were hampered by the restrictions, which conventional milling techniques impose. Modern rapid prototyping techniques, where layer by layer is added to the growing piece either by light driven polymerization or by three-dimensional printing techniques, overcome this constraint, allowing highly concave or even embedded and entangled structures. We have employed such a technique to manufacture an atomic force microscopy (AFM) head, and we compared its performance with a copy milled from aluminum. We tested both AFM heads for single molecule force spectroscopy applications and found little to no difference in the signal-to-noise ratio as well as in the thermal drift. The lower E modulus seems to be compensated by higher damping making this material well suited for low noise and low drift applications. Printing an AFM thus offers unparalleled freedom in the design and the rapid production of application-tailored custom instruments.     

Genetic Dissection of Light-Regulated Adventitious Root Induction in Arabidopsis thaliana Hypocotyls

Photomorphogenic responses of etiolated seedlings include the inhibition of hypocotyl elongation and opening of the apical hook. In addition, dark-grown seedlings respond to light by the formation of adventitious roots (AR) on the hypocotyl. How light signaling controls adventitious rooting is less well understood. Hereto, we analyzed adventitious rooting under different light conditions in wild type and photomorphogenesis mutants in Arabidopsis thaliana. Etiolation was not essential for AR formation but raised the competence to form AR under white and blue light. The blue light receptors CRY1 and PHOT1/PHOT2 are key elements contributing to the induction of AR formation in response to light. Furthermore, etiolation-controlled competence for AR formation depended on the COP9 signalosome, E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC (COP1), the COP1 interacting SUPPRESSOR OF PHYA-105 (SPA) kinase family members (SPA1,2 and 3) and Phytochrome-Interacting Factors (PIF). In contrast, ELONGATED HYPOCOTYL5 (HY5), suppressed AR formation. These findings provide a genetic framework that explains the high and low AR competence of Arabidopsis thaliana hypocotyls that were treated with dark, and light, respectively. We propose that light-induced auxin signal dissipation generates a transient auxin maximum that explains AR induction by a dark to light switch.     

Perinatal Obesity Induces Hepatic Growth Restriction with Increased DNA Damage Response,Senescence, and Dysregulated Igf-1-Akt-Foxo1 Signaling in Male Offspring of Obese Mice

Maternal obesity predisposes for hepato-metabolic disorders early in life. However, the underlying mechanisms causing early onset dysfunction of the liver and metabolism remain elusive. Since obesity is associated with subacute chronic inflammation and accelerated aging, we test the hypothesis whether maternal obesity induces aging processes in the developing liver and determines thereby hepatic growth. To this end, maternal obesity was induced with high-fat diet (HFD) in C57BL/6N mice and male offspring were studied at the end of the lactation [postnatal day 21 (P21)]. Maternal obesity induced an obese body composition with metabolic inflammation and a marked hepatic growth restriction in the male offspring at P21. Proteomic and molecular analyses revealed three interrelated mechanisms that might account for the impaired hepatic growth pattern, indicating prematurely induced aging processes: (1) Increased DNA damage response (γH2AX), (2) significant upregulation of hepatocellular senescence markers (Cdnk1a, Cdkn2a); and (3) inhibition of hepatic insulin/insulin-like growth factor (IGF)-1-AKT-p38-FoxO1 signaling with an insufficient proliferative growth response. In conclusion, our murine data demonstrate that perinatal obesity induces an obese body composition in male offspring with hepatic growth restriction through a possible premature hepatic aging that is indicated by a pathologic sequence of inflammation, DNA damage, senescence, and signs of a possibly insufficient regenerative capacity.     

Structural Analysis of Mitochondrial Dynamics—From Cardiomyocytes to Osteoblasts: A Critical Review

Mitochondria play a crucial role in cell physiology and pathophysiology. In this context, mitochondrial dynamics and, subsequently, mitochondrial ultrastructure have increasingly become hot topics in modern research, with a focus on mitochondrial fission and fusion. Thus, the dynamics of mitochondria in several diseases have been intensively investigated, especially with a view to developing new promising treatment options. However, the majority of recent studies are performed in highly energy-dependent tissues, such as cardiac, hepatic, and neuronal tissues. In contrast, publications on mitochondrial dynamics from the orthopedic or trauma fields are quite rare, even if there are common cellular mechanisms in cardiovascular and bone tissue, especially regarding bone infection. The present report summarizes the spectrum of mitochondrial alterations in the cardiovascular system and compares it to the state of knowledge in the musculoskeletal system. The present paper summarizes recent knowledge regarding mitochondrial dynamics and gives a short, but not exhaustive, overview of its regulation via fission and fusion. Furthermore, the article highlights hypoxia and its accompanying increased mitochondrial fission as a possible link between cardiac ischemia and inflammatory diseases of the bone, such as osteomyelitis. This opens new innovative perspectives not only for the understanding of cellular pathomechanisms in osteomyelitis but also for potential new treatment options.     

MRI-guided focused ultrasound: methodology and applications

Focused ultrasound is very well suited for inducing noninvasive local hyperthermia. Since magnetic resonance imaging (MRI) may be employed to obtain real-time temperature maps noninvasively the combination of these two technologies offers great advantages specifically aimed toward oncological studies. Real-time identification of the target region and accurate control of the temperature evolution during the treatment has now become possible. Thermal ablation of pathological tissue, local drug delivery using thermosensitive micro-carriers and controlled transgene expression using thermosensitive promoters have recently been demonstrated with this unique technology. Based on these experiments combined focused ultrasound and MRI thermometry holds promise for future oncological diagnostics and treatment. In this paper, we review some of the recent methodological developments as well as experimental and first clinical studies using this approach.     

A comprehensive approach to the analysis of contrast enhanced cardiac MR images

Current magnetic resonance imaging (MRI) technology allows the determination of patient-individual coronary tree structure, detection of infarctions, and assessment of myocardial perfusion. Joint inspection of these three aspects yields valuable information for therapy planning, e.g., through classification of myocardium into healthy tissue, regions showing a reversible hypoperfusion, and infarction with additional information on the corresponding supplying artery. Standard imaging protocols normally provide image data with different orientations, resolutions and coverages for each of the three aspects, which makes a direct comparison of analysis results difficult. The purpose of this work is to develop methods for the alignment and combined analysis of these images. The proposed approach is applied to 21 datasets of healthy and diseased patients from the clinical routine. The evaluation shows that, despite limitations due to typical MRI artifacts, combined inspection is feasible and can yield clinically useful information.     

A safe transmission line for MRI

Magnetic resonance imaging (MRI) has been established as a reliable and safe imaging method for the human body. However, electric conductors, such as cables situated near or in the human body, should be avoided because induced currents in the cables can cause hazardous heating in the surrounding tissue. In this paper, a new principle for the design of a transmission line is introduced and demonstrated, which is capable of avoiding dangerous heating of cables. The principle is based on transformers placed along the line, splitting the long line into several short not resonant and thus safe sections. A transformer design is introduced along with the theoretical aspects for both the avoidance of the undesired induced currents and the reduction of signal attenuation. Furthermore, the design fulfills the geometrical requirements of the side lumen of a standard catheter. Matching networks, whose elements are determined by power matching, are used to reduce signal attenuation by the transformers. A prototype was built to validate both theory and the simulations. As demonstrated in this work, it is possible to build safe transmission lines for MRI, making applications such as active catheter tracking possible. We expect that even new applications, such as safe intravascular imaging will be possible in a safe manner in the future.     

Compensation of sound speed deviations in 3-D B-mode ultrasound for intraoperative determination of the anterior pelvic plane

An accurate determination of the pelvic orientation is inevitable for the correct cup prosthesis placement of navigated total hip arthroplasties. Conventionally, this step is accomplished by percutaneous palpation of anatomic landmarks. Sterility issues and an increased landmark localization error for obese patients lead to the application of B-mode ultrasound imaging in the field of computer-assisted orthopedic surgery. Many approaches have been proposed in the literature to replace the percutaneous digitization by 3-D B-mode ultrasound imaging. However, the correct depth localization of the pelvic landmarks could be significantly affected by the acoustic properties of the penetrated tissues. Imprecise depth estimation could lead to a miscalculation of the pelvic orientation and subsequently to a misalignment of the acetabular cup implant. But so far, no solution has been presented, which compensates for acoustic property differences for correct depth estimation. In this paper, we present a novel approach to determine pelvic orientation from ultrasound images by applying a hierarchical registration scheme based on patch statistical shape models to compensate for differences in speed of sound. The method was validated based on plastic bones and a cadaveric specimen.